WO2022057862A1 - Anti-integrin antibody or antigen-binding fragment, and use thereof - Google Patents

Abstract

Provided is an anti-integrin antibody or antigen-binding fragment. The antibody or antigen-binding fragment specifically binds to αvβ8. Also provided is the use of the antibody or antigen-binding fragment in the preparation of a drug for treating or ameliorating αvβ8-related diseases, or the use thereof in the preparation of a kit for diagnosing αvβ8-related diseases.

Description

Anti-integrin antibody or antigen-binding fragment and application thereof technical field

The invention belongs to the field of biotechnology, and particularly relates to anti-αvβ8 antibodies or antigen-binding fragments and applications thereof.

Background technique

The multifunctional cytokine transforming growth factor beta (TGFβ) superfamily proteins are widely involved in a variety of biological and physiological processes, and have important biological functions on endothelial cells, mesenchymal cells, epithelial cells and immune cells. TGFβ generally binds non-covalently to the propeptide (LAP) and remains in an inactive state. This propeptide is referred to as the latency associated domain (LAP) of TGFβ. For TGFβ to function, it must first undergo an activation process and be released from the inactive complex. The complex typically contains: active TGF[beta] dimer, LAP and LTBP (latent TGF[beta] binding protein such as GARP).

There are 3 different isoforms of TGFβ, TGFβ1, 2 and 3, respectively. For the immune system, TGFβ regulates the function of regulatory T cells and the homeostasis of immune precursor cells. Regarding ECM remodeling, TGFβ signaling can promote fibroblast populations and ECM aggregation. In the tumor microenvironment, due to physiological dysregulation, TGFβ signaling promotes tumor angiogenesis, alters the stromal environment, and suppresses the activity of the immune system. In addition, enhanced TGFβ signaling can lead to diseases such as cardiovascular stenosis and glomerulosclerosis.

Integrins are cell surface receptors that generally contain two non-covalently bound α subunits and β subunits, mainly involved in cell-to-cell and cell-to-extracellular matrix (ECM) adhesion and signaling Conduction provides adhesion and other functions for cell growth, migration and differentiation during organ development and tissue damage. The binding specificity of integrins is generally determined by α subunits and β subunits. At present, 18 kinds of α subunits and 8 kinds of β subunits have been identified, a total of 24 different combinations of α subunits and β subunits (FGGiancotti et al. , et.al., Science, 285, 1028-1032, 1999).

αvβ8 is a member of the integrin superfamily. αvβ8 can bind to L-TGFβ, so that TGFβ is released from its precursor (Latent TGF-beta, L-TGFβ), resulting in the mature activation of TGFβ1 and TGFβ3 (Mu et al. (2002) J.Cell Biol.159: 493). αvβ8 is distributed in epithelial cells, neuronal tissues and mesenchymal cells. Moreover, Treg cells can activate TGFβ by expressing αvβ8, thereby inhibiting immune activity.

In conclusion, it is necessary to provide an antibody that, by binding to αvβ8, blocks the binding of αvβ8 to L-TGFβ, thereby locally inhibiting TGFβ signaling, which can be used to effectively and safely treat diseases and disorders involving TGFβ , including, for example, cancer, fibrosis and inflammation.

SUMMARY OF THE INVENTION

The present invention provides antibodies or antigen-binding fragments that specifically bind to αvβ8. In some embodiments, the antibodies or antigen-binding fragments provided herein are isolated antibodies or antigen-binding fragments. In some embodiments, the present invention provides antibodies or antigen-binding fragments that specifically bind human αvβ8 or monkey αvβ8. After the anti-αvβ8 antibody of the present invention specifically binds to αvβ8, it can block the combination of αvβ8 and L-TGFβ, thereby locally inhibiting the signal of TGFβ, reducing side effects and treating diseases related to TGFβ signal.

In some embodiments, the antibodies or antigen-binding fragments provided by the invention comprise:

(a) VH CDR1, it comprises the amino acid sequence shown in SEQ ID NO:5,

(b) VH CDR2, it comprises the amino acid sequence shown in SEQ ID NO:6,

(c) VH CDR3, it comprises the amino acid sequence shown in SEQ ID NO:7,

(d) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:44,

(e) a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 45, and/or

(f) VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:46.

In some embodiments, the VH CDR3 consists of 10-20 amino acids and comprises the fragment RGDL therein.

In some embodiments, the VH CDR3 comprises any of the amino acid sequences set forth in SEQ ID NOs: 8-25.

In some embodiments, the VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 12 or 14.

In some embodiments, the antibody or antigen-binding fragment specifically binds αvβ8, the antibody or antigen-binding fragment comprising:

(a) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, and/or

(b) a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:6, and/or

(c) a VH CDR3 comprising any of the amino acid sequences set forth in SEQ ID NOs: 8-25, and/or

(d) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 44, and/or

(e) a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 45, and/or

(f) VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment specifically binds αvβ8, the antibody or antigen-binding fragment comprising:

(a) VH CDR1, it comprises the amino acid sequence shown in SEQ ID NO:5,

(b) VH CDR2, it comprises the amino acid sequence shown in SEQ ID NO:6,

(c) a VH CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs: 8-25,

(d) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:44,

(e) a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and

(f) VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDRl set forth in SEQ ID NO:5, a VH CDRl set forth in SEQ ID NO:6, a VH set forth in SEQ ID NO:12 or 14 CDR3, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:8, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:9, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:10, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:11, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:13, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:15, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:16, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:17, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:18, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:19, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:20, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:21, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:22, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:23, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:24, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:25, VL CDR1 as set forth in SEQ ID NO:44, VL CDR2 as set forth in SEQ ID NO:45, and VL CDR3 as set forth in SEQ ID NO:46.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises any of the amino acid sequences set forth in SEQ ID NOs: 26-43, or a combination of any of the amino acid sequences set forth in SEQ ID NOs: 26-43 An amino acid sequence with at least 90% sequence homology, or an amino acid sequence with one or more conservative amino acid substitutions compared to any of the amino acid sequences shown in SEQ ID NOs: 26-43.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 30 or 32, or is at least 90 different from the amino acid sequence set forth in SEQ ID NO: 30 or 32 An amino acid sequence with % sequence homology, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 30 or 32.

In some embodiments, the light chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:4, or has at least 90% sequence homology to the amino acid sequence set forth in SEQ ID NO:4 The amino acid sequence of SEQ ID NO: 4 has one or more conservative amino acid substitutions compared with the amino acid sequence shown in SEQ ID NO: 4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:30, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:32, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:26, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:27 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:29 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:31 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:33, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:34, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:35, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:36 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:37, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:38 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:39 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:40, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:41 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:42, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:43 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4.

In some embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a kappa or lambda chain constant region. In some embodiments, the antibody or fragment thereof is one of the isotypes of IgG, IgM, IgA, IgE, or IgD. In some embodiments, the isotype is IgGl, IgG2, IgG3, or IgG4. In some embodiments, the isotype is IgGl. In some embodiments, the antibody or antigen-binding fragment is a murine, chimeric, or humanized antibody.

In some embodiments, the Fc is a variant Fc region. In some embodiments, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions or insertions, relative to the parent Fc region. In some embodiments, the amino acid modification of the Fc region alters effector function activity relative to the activity of the parental Fc region. In some embodiments, variant Fc regions may have altered (ie, increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding . In some embodiments, the Fc region amino acid modifications can alter the affinity of the variant Fc region for FcγRs (Fcγ receptors) relative to the parent Fc region. In some embodiments, the Fc region is derived from IgGl.

In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is scFV, Fab, F(ab)2, or IgGl. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.

In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:47, or has at least 90% sequence homology with the amino acid sequence set forth in SEQ ID NO:47 The amino acid sequence of SEQ ID NO:47, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 47; and/or the light chain constant region of the antibody or antigen-binding fragment comprises SEQ ID The amino acid sequence shown in NO: 48, or an amino acid sequence with at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 48, or an amino acid sequence with one or more homology compared with the amino acid sequence shown in SEQ ID NO: 48 Amino acid sequence of multiple conservative amino acid substitutions.

In some embodiments, the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:47, and the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:48 amino acid sequence.

In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 49 or 50, or is at least 90% identical in sequence to the amino acid sequence set forth in SEQ ID NO: 49 or 50 The derived amino acid sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO: 49 or 50; and/or the light chain of the antibody or antigen-binding fragment comprises SEQ ID The amino acid sequence shown in NO: 3, or the amino acid sequence with at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 3, or compared with the amino acid sequence shown in SEQ ID NO: 3, it has one or more Amino acid sequence of multiple conservative amino acid substitutions.

In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:49 and the light chain of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:3.

In some embodiments, the heavy chain of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:50, and the light chain of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:3.

In some embodiments, the antibody or antigen-binding fragment specifically binds αvβ3. In some embodiments, the antibody or antigen-binding fragment specifically binds αvβ8 and αvβ3. In some embodiments, the antibody or antigen-binding fragment specifically binds αvβ8, αvβ6, and αvβ3.

In some embodiments, the antibody or antigen-binding fragment has an affinity value K _D < 100 nM for αvβ8. In some embodiments, the antibody or antigen-binding fragment has an affinity numerical K _D < 30 nM for αvβ8. In some embodiments, the antibody or antigen-binding fragment has an affinity value K _D < 100 nM for αvβ3. In some embodiments, the antibody or antigen-binding fragment has an affinity value K _D < 30 nM for αvβ3. In some embodiments, the antibody or antigen-binding fragment has an affinity value K _{D < 100 nM for αvβ8 and an affinity value K D <} 100 nM for αvβ3. In some embodiments, the antibody or antigen-binding fragment has an affinity value K _{D < 30 nM for αvβ8 and an affinity value K D <} 30 nM for αvβ3. In some embodiments, the antibody or antigen-binding fragment has an affinity value K _{D < 26.3 nM for αvβ8 and an affinity value K D <} 22 nM for αvβ3. In some embodiments, the antibody or antigen-binding fragment has an affinity value K _{D ≤ 14.6 nM for αvβ8 and an affinity value K D ≤} 5.18 nM for αvβ3. In some embodiments, the antibody or antigen-binding fragment has an affinity value K _D ≤ 14.6 nM for αvβ8, an affinity value K _D ≦31.9 nM for αvβ6, and a K _D value for αvβ3 ≦5.18 nM.

In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment.

In another aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment. In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

In another aspect, the present invention also provides a vector or host cell comprising the above-mentioned nucleic acid molecule. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cells are CHO cells, HEK cells (eg, HEK293F cells), BHK cells, Cos1 cells, Cos7 cells, CV1 cells, or murine L cells.

In another aspect, the present invention provides a composition comprising the above-mentioned antibody or antigen-binding fragment, and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides methods of making the antibodies or antigen-binding fragments described herein, comprising culturing the above-described host cells in a culture medium to produce the antibodies or antigen-binding fragments.

On the other hand, the present invention provides the application of the above-mentioned antibody or antigen-binding fragment or the above-mentioned composition in the preparation of a medicine for treating or improving TGFβ-related diseases, or in the preparation of a kit for diagnosing TGFβ-related diseases application.

In some embodiments, the TGF[beta]-related disease comprises cancer or an autoimmune disease.

In another aspect, the present invention also provides a method of treating or ameliorating a TGF[beta]-related disease in a patient in need thereof, the method comprising administering to the patient an effective dose of the above-described antibody or antigen-binding fragment.

In some embodiments, the method further comprises administering to the patient one or more therapeutic agents.

The antibody or antigen-binding fragment thereof provided by the present invention can specifically recognize and bind αvβ8, block the combination of αvβ8 and L-TGFβ, thereby locally inhibiting the signal of TGFβ, while reducing side effects, and treating diseases related to TGFβ signal.

Description of drawings

Figure 1 shows the binding of yeast antibody clones to αvβ8 antigen.

Figure 2 shows the binding of the anti-αvβ8 antibody to the αvβ8 antigen on the surface of CHO cells.

Fig. 3 is an experiment of blocking the binding of αvβ8 to its ligand L-TGFβ by anti-αvβ8 antibody.

Figure 4 shows the results of cell biological activity detection of anti-αvβ8 antibodies.

detailed description

definition

Unless otherwise specified, each of the following terms shall have the meaning set forth below.

Where there are two or more definitions of a term used and/or accepted in the art, the definition of the term used herein includes all such meanings unless explicitly stated to the contrary.

"About" refers to the conventional error range of the corresponding numerical value readily known to those skilled in the relevant art. In some embodiments, references herein to "about" refer to the recited value and ranges of ±10%, ±5%, or ±1% thereof.

It should be noted that the term "an" entity refers to one or more of such entities, eg "an antibody" should be understood to mean one or more antibodies, thus the term "an" (or "an" ), "one or more" and "at least one" are used interchangeably herein.

The terms "comprising" or "comprising" as used herein mean that the compositions, methods and the like include the recited elements, such as components or steps, but do not exclude others. "Consisting essentially of" means that the compositions and methods exclude other elements that have an essential effect on the characteristics of the combination, but do not exclude elements that have no essential effect on the compositions or methods. "Consisting of" means excluding elements not specifically recited

The term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptide" and refers to a molecule formed from monomers (amino acids) linked linearly by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single chain or chains of two or more amino acids, and does not refer to a particular length of the product. Thus, the definition of "polypeptide" includes a peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain" or any other term used to refer to two or more amino acid chains, and the term "polypeptide" may Used in place of, or used interchangeably with, any of the above terms. The term "polypeptide" is also intended to refer to the product of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage or non-native Amino acid modifications that occur. Polypeptides may be derived from natural biological sources or produced by recombinant techniques, but need not necessarily be translated from a given nucleic acid sequence. It can be produced by any means including chemical synthesis.

"Amino acid" refers to compounds containing both amino and carboxyl functional groups, such as alpha-amino acids. Two or more amino acids can form polypeptides through amide bonds (also known as peptide bonds). A single amino acid is encoded by a nucleic acid consisting of three nucleotides, so-called codons or base triplets. Each amino acid is encoded by at least one codon. The same amino acid is encoded by different codons called "degeneracy of the genetic code". Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine Amino acid (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I) ), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

"Conservative amino acid substitution" refers to the replacement of one amino acid residue by another amino acid residue containing a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein. Examples of amino acid classes containing chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) Basic side chains: lysine, Arginine and histidine; 6) Acidic side chains: aspartic acid and glutamic acid. The number of amino acids of "light chain variable region, heavy chain variable region, light chain and heavy chain conservative amino acid substitution" in the present invention is about 1, about 2, about 3, about 4, about 7, about 9, about 11, about 20, about 22, about 24, about 28, about 31, about 33, about 36, about 39, about 43, about 45 conservative amino acid substitutions , or a range between any two of these values (including endpoints) or any value therein.

The term "isolated" as used herein with reference to cells, nucleic acids, polypeptides, antibodies, etc., eg, "isolated" DNA, RNA, polypeptides, antibodies, refers to other components in the cell's natural environment, such as DNA or RNA, respectively of one or more of the isolated molecules. The term "isolated" as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material or cell culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not, and would not exist in, their natural state. The term "isolated" is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc. are typically prepared by at least one purification step. In some embodiments, the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% pure, or any of these values The range between any two values of , including the endpoint, or any value therein.

In the present invention, the term "recombinant" refers to polypeptides or polynucleotides, meaning forms of polypeptides or polynucleotides that do not exist in nature, which can be combined to produce polynucleotides or polypeptides that do not normally exist. .

"Homology" or "identity" or "similarity" refers to the sequence similarity between two polypeptides or between two nucleic acid molecules. Homology is determined by comparing the positions in each sequence that can be aligned. A molecule is homologous when a position in the sequences being compared is occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences.

"At least 90% sequence homology" is about 90% homology, about 91% homology, about 92% homology, about 94% homology, about 95% homology, about 98% homology homology, about 99% homology, or a range (including endpoints) between any two of these values, or any value therein.

A polynucleotide or polynucleotide region (or polypeptide or antibody sequence) having a certain percentage (eg, 90%, 95%, 98%, or 99%) of "sequence identity" or "sequence identity" to another sequence "Homology" refers to the percentage of bases (or amino acids) that are identical in the two sequences being compared when the sequences are aligned. The alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007). In some embodiments, the alignment is performed using default parameters. One such alignment program is BLAST with default parameters. Biologically equivalent polynucleotides are polynucleotides that have the above-specified percentages of homology and encode polypeptides having the same or similar biological activity.

A polynucleotide is a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when polynucleotides For RNA, thymine is replaced by uracil (U). A "polynucleotide sequence" can be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, eg for functional genomics and homology searches.

The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. For example: genes or gene fragments (e.g. probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA , recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence and, isolated RNA of any sequence. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. Structural modifications to nucleotides can be performed before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any polynucleotide of the present disclosure includes the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

The term "encoding" when applied to a polynucleotide refers to a polynucleotide referred to as "encoding" a polypeptide which, in its natural state or when manipulated by methods well known to those skilled in the art, can be produced by transcription and/or translation the polypeptide and/or fragments thereof.

In the present invention, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The antibody can be an intact antibody or any antigen-binding fragment thereof or a single chain thereof. The term "antibody" thus includes proteins or peptides in the molecule that contain part or the whole of a biologically active immunoglobulin molecule that binds to an antigen. Including but not limited to the complementarity determining region (CDR), heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain Chain constant region (CL), framework region (FR) or any part thereof. The CDR regions include the CDR regions of the light chain (VL CDR1-3) and the CDR regions of the heavy chain (VH CDR1-3).

A "monoclonal antibody" (mAb) is an antibody made from the same immune cell, which is all clones of a single parental cell. Monoclonal antibodies can have monovalent affinity because they bind to the same epitope (the portion of the antigen recognized by the antibody). In contrast, polyclonal antibodies bind to multiple epitopes and are typically secreted by several different plasma cells. Monoclonal antibodies can be prepared by hybridoma, recombinant, transgenic, or other techniques known to those of skill in the art.

Classes of antibody heavy chains include gamma, mu, alpha, delta, epsilon, and some subclasses (eg, gamma1-gamma4). The nature of this chain determines the "class" of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Some of these can be further divided into immunoglobulin subclasses (isotypes) such as IgGl, IgG2, IgG3, IgG4, etc. Classes of light chains include kappa, lambda. Each heavy chain can bind to a kappa or lambda light chain. Generally, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, their light and heavy chains are joined by covalent bonds, and the "tail" portion of the two heavy chains is joined by a covalent disulfide bond or non-covalent bond. In heavy chains, the amino acid sequence extends from the N-terminus of the Y-shaped fork-end to the C-terminus at the bottom of each chain. The variable region of immunoglobulin kappa light chain is Vκ; the variable region of immunoglobulin λ light chain is Vλ. VL commonly used in the present invention is Vκ. Although some discussions are directed to the IgG class of immunoglobulin molecules, all immunoglobulin classes are within the scope of the present disclosure. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of about 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of about 53,000-70,000. Both light and heavy chains can be divided into regions of structural and functional homology.

The terms "constant" and "variable" are used according to function. In this regard, it is understood that VL and VH determine antigen recognition and specificity. The antigen-binding sites on VL and VH are capable of recognizing antigenic determinants and binding antigen-specifically. The antigen binding site is defined by three CDRs each in VH and VL (i.e. VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3). CL and CH (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement fixation and the like. By convention, the numbering of constant regions increases as they become further from the antigen binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chains, respectively.

In the present invention, the heavy chain constant regions of the antibodies can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of an antibody can include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In some embodiments, the heavy chain constant region may include a hinge region derived in part from an IgGl molecule and in part from an IgG3 molecule. In some embodiments, a portion of the heavy chain may include a chimeric hinge region that is partially derived from an IgGl molecule and partially derived from an IgG4 molecule.

In the present invention, the term "hinge region" includes the part of the heavy chain structure connecting the CH1 domain and the CH2 domain. The hinge region comprises about 25 residues and is resilient, allowing the two N-terminal antigen binding regions to move independently.

In the present invention, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. Cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are connected by a disulfide bond, and the two heavy chains are connected by two disulfide bonds.

In the present invention, the term "fragment", "antibody fragment" or "antigen-binding fragment" is part of an antibody, eg, F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, and the like. Regardless of their structure, antibody fragments bind to the same antigen that is recognized by the intact antibody. The term "antigen-binding fragment" includes aptamers, Spiegelmers, and diabodies, as well as any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

"Single-chain variable fragment" or "scFv" refers to a fusion protein of the VH and VL of an immunoglobulin. In some aspects, these regions are linked to a short linker peptide of about 10 to about 25 amino acids. Linkers can be rich in glycine to increase flexibility, and serine or threonine to increase solubility, and can link the N-terminus of VH and the C-terminus of VL, and vice versa. Although the constant region has been removed and the linker introduced, the protein retains the specificity of the original immunoglobulin.

Antibodies, antigen-binding fragments, variants or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, or chimeric antibodies, single chain antibodies , epitope-binding fragments.

The term "epitope" as used herein includes any protein-determining region capable of specifically binding an immunoglobulin or fragment thereof or a T cell receptor. Epitope-determining regions usually consist of chemically active surface groups of molecules (eg, amino acids or sugar side chains) and usually have specific three-dimensional structural properties as well as specific charge properties. When the dissociation constant is less than or equal to 1 μM (eg, less than or equal to 100 nM, less than or equal to 10 nM, or less than or equal to 1 nM), the antibody can be said to specifically bind to the antigen.

CDRs as defined by Kabat and Chothia include overlaps or subsets of amino acid residues when compared to each other. Nonetheless, it is within the scope of the invention to apply either definition to refer to the CDRs of an antibody or variant thereof. The exact residue numbers encompassing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can usually determine which specific residues the CDRs contain based on the amino acid sequence of the variable region of the antibody.

Kabat et al. also define a numbering system applicable to variable region sequences of any antibody. One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence independent of experimental data other than the sequence itself. "Kabat Numbering" means the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies may also use the EU numbering system.

The antibodies disclosed herein can be derived from any animal, including birds and mammals. Preferably, the antibody is of human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken origin. In some embodiments, the variable regions may be of chondrichthyes origin (eg, from sharks).

In the present invention, the term "chimeric antibody" is taken to mean that the variable regions of the antibody are obtained or derived from a first species, while the constant regions thereof (which may be complete, partial or modified in the present invention) ) any antibody derived from a second species. In some embodiments, the variable regions are of non-human origin (eg, mouse or primate), and the constant regions are of human origin.

"Specifically binds" generally refers to the formation of a relatively stable complex of an antibody or antigen-binding fragment with a specific antigen through complementary binding of its antigen-binding domain to an epitope. "Specificity" can be expressed in terms of the relative affinity with which an antibody or antigen-binding fragment binds to a particular antigen or epitope. For example, if antibody "A" has a greater relative affinity for the same antigen than antibody "B", antibody "A" can be considered to be more specific for that antigen than antibody "B". Specific binding can be described by the equilibrium dissociation constant ( _KD ), with a smaller KD _implying tighter binding. Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, optical interferometry of biological film layers, and the like. Antibodies that "specifically bind" the αvβ8 protein include those with an equilibrium dissociation constant KD of less than or equal to about 30 nM, less than or equal to about 26 nM, or less than or equal to about 15 nM with the αvβ8 protein.

"Treatment" means therapeutic treatment and prophylactic or prophylactic measures, the purpose of which is to prevent, slow, ameliorate, or stop an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following, whether detectable or undetectable As a result, remission of symptoms, reduction of disease severity, stabilization of disease state (ie, no worsening), delay or slowdown of disease progression, improvement, alleviation, alleviation or disappearance of disease state (whether in part or in whole), prolongation and Expected duration of survival when not receiving treatment, etc. Patients in need of treatment include patients already suffering from a condition or disorder, a patient susceptible to a condition or disorder, or a patient in need of prevention of such a condition or disorder, may or may be expected from administration of the antibodies or pharmaceutical compositions disclosed herein for detection , patients who benefit from the diagnostic process and/or treatment.

"Patient" refers to any mammal in need of diagnosis, prognosis, or treatment, including humans, dogs, cats, rabbits, mice, horses, cows, and the like.

In the present invention, references such as "patients in need of treatment" include patients, eg mammalian patients, who would benefit from administration of the antibodies or compositions disclosed herein for detection, diagnostic procedures, prophylaxis and/or treatment.

The term "cytokine" is a generic term for proteins released by one cell population that act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, interleukins (IL) (such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7. IL-8, IL-9, IL-11, IL-12, IL-15), tumor necrosis factor (such as TNF-α or TNF-β) and other polypeptide factors (including LIF and kit ligand (KL) and gamma-interferon). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native sequence cytokines. Biologically active equivalents include synthetically produced small molecular entities, and pharmaceutically acceptable derivatives and salts thereof.

As used herein, the term "label" or "labeled" refers to the incorporation of a detectable label, eg, by incorporation of a radiolabeled amino acid, or attachment to a labelable avidin (eg, containing a fluorescent label or readable by optical A polypeptide with a biotinyl moiety detected by the method or calorimetrically having an enzymatically active streptavidin). In certain instances, the marker or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (eg ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I) , fluorescent labels (eg FITC, rhodamine, lanthanide phosphors), enzymatic labels (eg horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biological acyl groups, predetermined polypeptide epitopes recognized by secondary reporter genes (eg, leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags).

Anti-αvβ8 antibody

The antibodies of the present invention have the ability to bind to αvβ8. In some embodiments, the antibodies of the invention have the ability to bind human αvβ8 or cynomolgus αvβ8.

In some embodiments, the antibody αvβ8 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH), wherein the VH comprises the complementarity determining regions VH CDR1, VH CDR2 and VH CDR3. wherein VH CDR1 comprises an amino acid sequence having at least 50%, 60%, 70%, 80% or 90% identity or 100% identity to the amino acid sequence shown in SEQ ID NO: 5, and VH CDR2 comprises an amino acid sequence with SEQ ID NO: The amino acid sequence shown in 6 has an amino acid sequence of at least 50%, 60%, 70%, 80% or 90% identity or 100% identity, and the VH CDR3 comprises an amino acid sequence shown in SEQ ID NO: 7 with at least Amino acid sequences of 50%, 60%, 70%, 80% or 90% identity or 100% identity.

In some embodiments, the VH CDR3 contains 10-20 amino acids comprising the sequence RGDL (SEQ ID NO:7). In some embodiments, the VH CDR3 contains 16-18 amino acids comprising the sequence RGDL. In some embodiments, the VH CDR3 comprises any of the amino acid sequences set forth in SEQ ID NOs: 8-25. In some embodiments, the VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO:14.

In some embodiments, the anti-αvβ8 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL), wherein the VL comprises a complementarity determining region (CDR) VL CDR1, VL CDR2 and VL CDR3, wherein VL CDR1 Comprising an amino acid sequence having at least 50%, 60%, 70%, 80% or 90% identity or 100% identity with the amino acid sequence shown in SEQ ID NO:44, VL.CDR2 comprises the amino acid sequence shown in SEQ ID NO:45 The amino acid sequence shown has at least 50%, 60%, 70%, 80% or 90% identity or an amino acid sequence with 100% identity, and the VL CDR3 comprises at least 50%, Amino acid sequences of 60%, 70%, 80% or 90% identity or 100% identity.

In some embodiments, the VH CDR1 of the anti-αvβ8 antibody or antigen-binding fragment thereof of the invention comprises the amino acid sequence shown in SEQ ID NO:5, the VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:6, the VH CDR3 comprise the amino acid sequence shown in SEQ ID NO:12; VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:44, VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:45, and VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:45 : amino acid sequence shown in 46.

In some embodiments, the VH CDR1 of the antibody αvβ8 antibody or antigen-binding fragment thereof of the invention comprises the amino acid sequence set forth in SEQ ID NO:5, the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO:6, the VH CDR3 Comprising the amino acid sequence shown in SEQ ID NO:14; VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:45 : amino acid sequence shown in 46.

The CDRs in the antibodies of the present invention or antigen-binding fragments thereof can be any exemplary combination of amino acid sequences corresponding to each of the CDRs in Table 1.

Table 1 CDR sequences

SEQ ID NO	CDRs	sequence
5	VH CDR1		SYAMS
6	VH CDR2		AISGSGGSTYYADSVKG
8	VH CDR3		AGRVGLRGDLPPGTPVDY
9	VH CDR3		TGVATGRGDLGAHGRIDY
10	VH CDR3	TRQNALRGDLQPRFTSDY
11	VH CDR3		WMQSRGDLPAWASSDY
12	VH CDR3		DRHWAVRGDLAVLQPKDY
13	VH CDR3		EMFNGRRGDLPQGPRHDY
14	VH CDR3	SALLPSRGDLPLRWVSDY
15	VH CDR3		IATLVRGDLGWLHENDY
16	VH CDR3	GNMFPSRGDLSPLAVADY

17	VH CDR3	ILSGRGDLGWLSSPDY
18	VH CDR3	ILLFFDRGDLPRGHLFDY
19	VH CDR3		VLPGRGDLPHWTLSDY
20	VH CDR3	LGHAQRGDLPRNSDLDY
twenty one	VH CDR3	SVVTSKRGDLAGQPVRDY
twenty two	VH CDR3	TSPARGDLPALRTSDY
twenty three	VH CDR3	PLASRGDLPSFASSDY
twenty four	VH CDR3		SPFFHTRGDLASSYLSDY
25	VH CDR3	TPPARGDLPNLALSDY
44	VL CDR1	RASQGISSYLA
45	VL CDR2	AASSLQS
46	VL CDR3	QQHYTTPPT

In some embodiments, the anti-αvβ8 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region VL comprising at least 90%, 91%, 92%, 93%, and 93% of the amino acid sequence set forth in SEQ ID NO:4 %, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical amino acid sequences.

In some embodiments, the anti-αvβ8 antibody or fragment thereof of the invention comprises a heavy chain variable region VH comprising at least 90%, 91%, 92%, 93% with any of the amino acid sequences of SEQ ID NOs: 26-43 %, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical amino acid sequences.

In some embodiments, the antibodies of the invention comprise a heavy chain constant region (CH) as set forth in SEQ ID NO:47, and a light chain constant region (CL) as set forth in SEQ ID NO:48.

In some embodiments, Antibody 1 comprises VH as set forth in SEQ ID NO:26, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, Antibody 2 comprises VH as set forth in SEQ ID NO:27, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, Antibody 3 comprises VH as set forth in SEQ ID NO:28, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, Antibody 4 comprises VH as set forth in SEQ ID NO:29, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, Antibody 9 comprises VH as set forth in SEQ ID NO:30, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, Antibody 13 comprises VH as set forth in SEQ ID NO:31, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, antibody 16 comprises VH as set forth in SEQ ID NO:32, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, antibody 21 comprises VH as set forth in SEQ ID NO:33, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, antibody 22 comprises VH as set forth in SEQ ID NO:34, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, antibody 23 comprises VH as set forth in SEQ ID NO:35, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, antibody 24 comprises VH as set forth in SEQ ID NO:36, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, antibody 25 comprises VH as set forth in SEQ ID NO:37, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, antibody 27 comprises VH as set forth in SEQ ID NO:38, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, antibody 28 comprises VH as set forth in SEQ ID NO:39, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, antibody 29 comprises VH as set forth in SEQ ID NO:40, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, antibody 30 comprises VH as set forth in SEQ ID NO:41, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, antibody 31 comprises VH as set forth in SEQ ID NO:42, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, antibody 32 comprises VH as set forth in SEQ ID NO:43, VL as set forth in SEQ ID NO:4, CH as set forth in SEQ ID NO:47, as set forth in SEQ ID NO:48 CL.

In some embodiments, anti-αvβ8 antibodies, antigen-binding fragments, variants or derivatives are disclosed. A variant refers to an antibody or antigen-binding fragment thereof obtained by deleting and/or replacing one or more amino acid residues in an antibody or an antigen-binding fragment thereof, or inserting one or more amino acid residues. Derivatives include derivatives that are modified, ie, modified by covalent attachment of any type of molecule to the antibody, wherein the covalent attachment does not prevent the antibody from binding to the epitope. Including but not limited to the following examples, antibodies can be linked to cellular ligands by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or other proteins, etc. Any of a number of chemical modifications can be performed by existing techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, antibodies may contain one or more unnatural amino acids.

In some embodiments, the antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

Antibodies can be conjugated or fused to therapeutic agents, which can include detectable labels, such as radiolabels, immunomodulatory agents, hormones, enzymes, oligonucleotides, photosensitizing therapeutic or diagnostic agents, which can be pharmaceutical or toxin Cytotoxic agents, ultrasound enhancers, non-radioactive labels and compositions thereof, and other such agents known in the art.

Antibodies can be detectably labeled by conjugating them to chemiluminescent compounds. The presence of the chemiluminescently labeled antigen-binding fragment is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

In some embodiments, the antibodies of the invention also encompass variants of the amino acid sequence of anti-αvβ8 antibodies, as well as antibodies that bind the same epitope as any of the antibodies described above.

In some embodiments, the anti-αvβ8 antibodies of the invention also encompass antibody fragments thereof; in some embodiments, antibody fragments selected from Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragments.

In some embodiments, the present invention provides nucleic acids encoding any of the above anti-αvβ8 antibodies or fragments thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. Expression vectors include plasmids, retroviruses, YAC, EBV-derived episomes, and the like. In one embodiment, a host cell comprising the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293F cells) or other cells suitable for the production of antibodies or antigen-binding fragments thereof.

In some embodiments, the nucleic acid sequence encoding the anti-αvβ8 antibody or fragment thereof can be obtained from the amino acid sequence of the anti-αvβ8 antibody or fragment thereof by methods routine in the art.

In some embodiments, the antibody is chimeric, humanized or fully human. In some embodiments, the antibody or fragment thereof activates T cells, promotes their proliferation or secretes inflammatory factors. In some embodiments, the antibody or fragment thereof is an IgG isotype selected from the group consisting of IgGl isotype, IgG2 isotype, IgG3 isotype and/or IgG4 isotype group. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG isotype selected from IgGl.

The invention also includes antibodies that bind the same epitope as the anti-αvβ8 antibodies described herein. For example, the antibodies of the invention specifically bind to an epitope that includes one or more amino acid residues on human αvβ8.

Those skilled in the art will recognize that it is possible to determine whether an antibody binds to the same epitope as an antibody described herein without undue experimentation by simply finding out whether the antibody to be tested prevents binding of a known antibody to αvβ8. If a test antibody competes with an antibody of the disclosure, then the two antibodies are likely to bind to the same or similar epitope.

An alternative method for determining whether an antibody has the specificity of the antibody described herein is to pre-incubate the antibody described herein with a soluble αvβ8 protein to which the antibody typically responds, and then add the antibody to be tested to determine the test Whether the ability of the antibody to bind to αvβ8 is inhibited. If the tested antibody is inhibited, it has the same epitope specificity, or the same function as the antibody of the present disclosure.

In some embodiments, the antibodies of the invention can be prepared using, for example, the methods described in the Examples provided below. In some embodiments, also by using Trioma technology, human B cell hybridoma technology (see Kozbor et al, 1983, Immunol Today 4:72), and EBV hybridoma technology (see Cole et al, 1985, In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) etc.

Using conventional recombinant DNA techniques, one or more CDRs of an antibody of the invention can be inserted into a framework region, eg, into a human framework region, to construct a humanized, non-fully human antibody. Framework regions can be naturally occurring or consensus framework regions, preferably human framework regions (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a list of human framework regions). Some polynucleotides may encode antibodies that produce combinations of framework regions and CDRs that specifically bind to at least one epitope of an antigen of interest. One or more amino acid substitutions may be made within the framework regions and may be selected to improve binding of the antibody to its antigen. In addition, substitution or deletion of cysteine residues in one or more variable regions involved in interchain disulfide bond formation can be performed using this method, resulting in antibody molecules lacking one or more interchain disulfide bonds. Other changes to polynucleotides that are within the skill in the art are also encompassed by the present invention.

In addition, antibodies can be prepared using conventional recombinant DNA techniques. Antibody-producing vectors and cell lines can be selected, constructed and cultured using recombinant DNA techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, DLHacker, FMWurm, in Reference Module in Life Sciences, 2017, which in their entirety include Supplementary content is incorporated by reference in its entirety.

In some embodiments, DNA encoding the antibody can be designed and synthesized according to the amino acid sequences of the antibodies described herein according to conventional methods, placed in an expression vector, then transfected into host cells, and the transfected host cells are grown in culture to produce antibody. An antibody expression vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence. Efficient transcription can be obtained by the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, and other cellular promoters such as muscle can be used. kinesin promoter. Suitable expression vectors may include pYD, pIRES1neo, pRetro-Off, pRetro-On, PLXSN, Plncx, pCHO1.0, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro( +/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2 etc. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells.

In some embodiments, the inserted gene fragment needs to contain a selectable marker. Common selectable markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening isolation of successful cells. The constructed plasmids are transfected into host cells and cultured in selective medium. The successfully transfected cells grow in large numbers to produce the desired target protein.

Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G, immunoaffinity chromatography, and the like. For example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (2000), pp. 25-28) discusses the purification of immunoglobulins.

In addition, the anti-αvβ8 antibody of the present invention can specifically bind to αvβ3 at the same time. There is now much evidence that progressive tumor growth is dependent on angiogenesis, the formation of new blood vessels that supply the tumor with nutrients and oxygen, carry away waste products and serve as conduits for distant metastasis (Gastl et al., Oncol. 54 : 177-184). Recent studies have further identified the role of integrins in the process of angiogenesis. Integrins are heterodimeric transmembrane proteins that play critical roles in cell adhesion to the extracellular matrix (ECM), mediating cell survival, proliferation and migration through intracellular signaling. During angiogenesis, many integrins expressed on the surface of activated endothelial cells regulate key adhesion interactions, and many ECM proteins regulate different biological events such as cell migration, proliferation, and differentiation. Integrin αVβ3 mediates an independent pathway during angiogenesis. Antibodies against αvβ3 block basic fibroblast growth factor (bFGF)-induced angiogenesis. αvβ3 can be expressed in a variety of malignant tumors of different tissue origins, including epithelial tumors, lymphocyte-derived tumors, and neuroectodermal-derived tumors represented by melanoma. And αvβ3 can promote tumor invasion and metastasis, inhibit tumor cell apoptosis. The anti-αvβ8 antibody of the present invention can bind to both integrin αvβ8 and integrin αvβ3. After the anti-αvβ8 antibody binds to the two targets, it exerts anti-tumor effects from different ways, which may further improve the anti-tumor activity of the antibody.

In addition to αvβ8, the integrin αvβ6 is also capable of binding LAP and also causes TGF-β to be released from its precursor (Latent TGF-beta), leading to the maturational activation of TGF-β1 and 3 (Mu et al (2002) J. Cell Biol. 159:493). In some embodiments, the anti-αvβ8 antibody provided by the present invention can have higher affinity with αvβ6 in addition to higher affinity with αvβ8, further exert the effect of the antibody to inhibit TGFβ signal transduction, more effectively and safely use For the treatment of diseases and disorders involving TGFβ, including, for example, cancer, fibrosis and inflammation.

Use of anti-αvβ8 antibodies or antigen-binding fragments thereof

In some embodiments, there is provided a method of treating or ameliorating a TGF[beta]-related disease in a patient in need thereof, the method comprising administering to the patient an effective dose of an antibody or antigen-binding fragment described herein. In some embodiments, use of an antibody or antigen-binding fragment herein in the manufacture of a medicament for the treatment or amelioration of a TGF[beta]-related disease is provided. Diseases or disorders that can be treated by the anti-αvβ8 antibodies described herein include hematological cancers and/or solid tumors.

Blood cancers include, for example, leukemia, lymphoma, and myeloma. In some embodiments, the leukemia comprises acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); ). Lymphomas include Hodgkin lymphoma, indolent and aggressive non-Hodgkin lymphoma, Burkitt lymphoma, and follicular lymphoma (small cell and large cell). Myeloma includes multiple myeloma (MM), giant cell myeloma, heavy chain myeloma and light chain or Bence-Jones myeloma. Solid tumors include, for example, breast, ovarian, lung, pancreatic, prostate, melanoma, colorectal, lung, head and neck, bladder, esophagus, liver, and kidney cancers.

In some embodiments, the antibodies of the invention can activate an immune response for use in the treatment of infections.

Infection is the invasion of organisms' tissues by pathogenic agents, their reproduction, and the response of host tissues to these organisms and the toxins they produce. Infections may be caused by infectious agents such as viruses, viroids, prions, bacteria, nematodes such as parasitic roundworms and pinworms, arthropods such as ticks, mites, fleas and lice, fungi such as ringworm, and other macroparasites such as tapeworms and other worm. In one aspect, the infectious agent is a bacterium, such as a Gram-negative bacterium. In one aspect, the infectious agent is a virus, such as a DNA virus, an RNA virus, and a retrovirus. Non-limiting examples of viruses include adenovirus, coxsackie virus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, human Herpes virus type 8, HIV, influenza virus, measles virus, mumps virus, human papilloma virus, parainfluenza virus, polio virus, rabies virus, respiratory syncytial virus, rubella virus, varicella-zoster virus.

The antibodies of the present invention can also be used to treat infectious diseases caused by microorganisms, or to eliminate microorganisms by targeting microorganisms and immune cells to kill microorganisms. In one aspect, the microorganism is a virus, including RNA and DNA viruses, Gram-positive bacteria, Gram-negative bacteria, protozoa, or fungi.

A therapeutically effective amount of an antibody of the invention relates to the amount required to achieve the therapeutic goal. The amount required for administration depends on the binding affinity of the antibody for its specific antigen, the severity of the disease, disorder or condition, the route of administration, the rate at which the administered antibody is depleted from free volume in the subject receiving it, and the like. In some embodiments, a therapeutically effective dose of an antibody or antibody fragment of the invention ranges from about 0.01 mg/kg to about 100 mg/kg. In some embodiments, a therapeutically effective dose of an antibody or antibody fragment of the invention ranges from about 0.1 mg/kg to about 30 mg/kg. Dosing frequency can range, for example, from twice a week or to once every three weeks.

In some embodiments, an anti-αvβ8 antibody is administered to a patient diagnosed with clinical symptoms associated with one or more of the foregoing diseases, including but not limited to cancer or other neoplastic disorders. Following diagnosis, the anti-αvβ8 antibody is administered to reduce or eliminate the effect of one or more of the aforementioned disease-related clinical symptoms.

Overexpression of αvβ8 has been observed in certain tumor samples, and patients with cells overexpressing αvβ8 and/or αvβ3 may respond to treatment with the anti-αvβ8 antibodies of the invention. Therefore, the antibodies of the present invention can also be used for diagnosis and prognosis.

In some embodiments, a sample comprising cells can be obtained from a patient, which can be a cancer patient or a patient to be diagnosed. Cells are cells of tumor tissue or tumor mass, blood sample, urine sample, or any sample from a patient. After selective pretreatment of the sample, the anti-αvβ8 antibody can be used to detect the αvβ8 protein in the sample by incubating the sample with an antibody of the invention under conditions that allow the antibody to interact with the αvβ8 protein that may be present in the sample The presence.

The antibodies of the present invention are also useful for the detection of αvβ8 in patient samples and are therefore useful in diagnosis. For example, the anti-αvβ8 antibodies of the invention are used in in vitro assays (eg, ELISA) to detect αvβ8 and/or αvβ3 levels in patient samples.

In one embodiment, the anti-αvβ8 antibodies of the invention are immobilized on a solid support such as the wells of a microtiter plate. The immobilized antibody acts as a capture antibody, capturing any αvβ8 that may be present in the test sample. Before contacting the immobilized antibody with the patient sample, the solid support is washed and treated with a blocking reagent such as milk protein or albumin to avoid nonspecific adsorption of the analyte. The wells are then treated with a test sample that may contain antigen or with a solution containing a standard amount of antigen. Such a sample is, for example, a serum sample from a subject, which may have circulating antigen levels believed to be diagnostic of a certain pathology. After washing away the test sample or standard, the solid support is treated with a detectably labeled secondary antibody. The labeled secondary antibody is used as the detection antibody. The level of detectable label is measured and the concentration of αvβ8 in the test sample is determined by comparison to a standard curve established with a standard sample.

In some embodiments, when using an anti-αvβ8 antibody or antigen-binding fragment thereof described herein, the antibody or fragment thereof is in the form of a pharmaceutical composition. Wherein, the pharmaceutical composition can be composed of anti-αvβ8 antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration . Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences. Such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and/or 5% human serum albumin.

In some embodiments, the pharmaceutical composition containing the anti-αvβ8 antibody is compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal. The pharmaceutical composition may include one or more of the following components: sterile diluents for injection, such as water, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; bacteriostatic agents, such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as histidine hydrochloride, acetate, Citrate or phosphate; osmotic pressure regulators such as sodium chloride or dextrose; stabilizers such as arginine, methionine, trehalose, sucrose, sorbitol; surfactants such as Tween 20 ,Tween 80. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Pharmaceutical compositions can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In some embodiments, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (herein water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In use, the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the antibody in the required amount in an appropriate solvent with one or more combinations of ingredients enumerated above, as required, followed by filter sterilization. Sterile solutions of the foregoing can also be obtained by freeze-drying to obtain powders for the preparation of sterile injectable solutions upon administration.

In some embodiments, the anti-αvβ8 antibody and other therapeutic agent are prepared as a single therapeutic composition, and the anti-αvβ8 antibody and other therapeutic agent are administered simultaneously. Alternatively, the anti-αvβ8 antibody and the other therapeutic agent are independent of each other, eg, prepared separately as separate therapeutic compositions and the anti-αvβ8 antibody and the other therapeutic agent are administered simultaneously, or at different times during the treatment regimen. For example, the anti-αvβ8 antibody is administered before the other therapeutic agent is administered, the anti-αvβ8 antibody is administered after the other therapeutic agent is administered, or the anti-αvβ8 antibody and the other therapeutic agent are administered on an alternating schedule. Herein, anti-αvβ8 antibodies and other therapeutic agents are administered in a single dose or in multiple doses.

Those skilled in the art will appreciate that the antibodies of the present invention have a variety of uses. For example, the antibodies of the invention can be used as therapeutics, as reagents in diagnostic kits or as diagnostic tools, or as reagents in competition experiments to generate therapeutics.

Example

Materials, reagents, etc. used in the following examples can be obtained from commercial sources or known methods unless otherwise specified.

Example 1: Production and purification of αvβ8 antigen and control antibody

1.1 Preparation of human αvβ8 antigen:

From the protein database Uniprot, find the amino acid sequences of the two subunits of human αvβ8 (ITGAV: P06756; ITGB8: P26012), wherein the amino acid sequences of the extracellular regions of the two subunits of human αvβ8, ITGAV and ITGB8, are 1 to 992, respectively. and residues 1 to 684. And at the C-terminus of the extracellular region of the two subunits, a long chain of acidic amino acids (SEQ IN NO: 1) and a long chain of basic amino acids (SEQ IN NO: 2) are respectively added. These two stretches of amino acids contribute to the stable assembly of the two subunits of ITGAV and ITGB8. They were inserted into pCHO1.0 plasmid (purchased from Invitrogen Company, A13696-01) by restriction enzyme digestion to obtain a recombinant plasmid. The above plasmids were then transiently transfected into HEK293 cells by PEI, and the supernatant was collected after 7 days of culture, and purified to obtain αvβ8-his protein samples, which were used in the following various examples.

1.2 Preparation of control antibodies C6D4 and 264RAD.

The variable region sequences of the heavy chain and light chain of antibody C6D4 are shown in 4F1F9 of patent WO2018064478, and the amino acid sequences of the constant regions of the heavy chain and light chain of antibody C6D4 are SEQ ID NO: 47 and SEQ ID NO: 48, respectively. The sequences of the heavy and light chains of antibody 264RAD are shown in patent CN103524619. The DNAs of the heavy chain and light chain were ligated into the pCHO1.0 plasmid by enzyme digestion and ligation, respectively, to obtain a recombinant plasmid for expressing the full antibody. Using the Freedom CHO-S kit (purchased from Invitrogen) according to the manufacturer's instructions, the above recombinant plasmids were transferred into the CHO-S cell line. After 11 days of culture, the supernatant was collected and purified to obtain C6D4 and 264RAD antibody protein samples. in the following various examples.

Example 2: Preparation of anti-human αvβ8 antibody

2.1 Antibody Fragment Fab Preparation

The VH of the antibody fragment Fab is shown in Table 2, wherein the underline is the CDR region; the VL is shown in SEQ ID NO: 4. The DNA encoding the above sequence was obtained by artificial synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd.), and the DNA of the heavy chain and the light chain were respectively connected to the yeast display plasmid pYD-VH-VL by PCR homologous recombination (the construction of the plasmid is referred to in Ref. Simon Rosowski,Stefan Becker,et al.A novel one-step approach for the construction of yeast surface display Fab antibody libraries.Microb.Cell Fact.(2018)17:3), and then electroporated into yeast to get a batch of and αvβ8 -His antigen-binding clone, the results of flow cytometry analysis of its binding to αvβ8-his antigen are shown in FIG. 1 . The sequencing result VH is shown in Table 2; VL is shown as SEQ ID NO:4.

SEQ ID NO: 4 is as follows (CDRs are underlined):

Table 2: Amino acid sequences of αvβ8-bound cloned heavy chain variable regions

2.2 Preparation of IgG whole antibody

IgG whole antibodies were prepared. The variable region of the heavy chain of the antibody is shown in Table 2, and the constant region of the antibody is of IgG1 type (the constant region of the heavy chain is shown in SEQ ID NO: 47, and the constant region of the light chain is shown in SEQ ID NO: 48). Design and synthesize the DNA fragments encoding the corresponding heavy and light chains of the above-mentioned antibodies, and ligate them into the pCHO1.0 plasmid by enzyme digestion to obtain a recombinant plasmid for expressing the full antibody. Using Freedom CHO-S kit (purchased from Invitrogen) according to the manufacturer's instructions, the above recombinant plasmids were transferred into CHO-S cell line, and after 11 days of culture, the supernatant was collected and purified by Protein A to obtain eighteen antibody strains.

The heavy chain constant region sequence SEQ ID NO: 47 is as follows:

The light chain constant region sequence SEQ ID NO: 48 is as follows:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The heavy chain sequence of antibody 9, SEQ ID NO: 49, is as follows (variable regions are underlined):

The heavy chain sequence of antibody 16, SEQ ID NO: 50, is as follows (variable regions are underlined):

The light chain sequence SEQ ID NO: 3 of each antibody is as follows (variable regions are underlined):

Example 3: Identification of the binding ability of anti-human αvβ8 antibody

3.1 The binding and specificity of anti-human αvβ8 antibody and αvβ8 were determined by ELISA.

The αvβ8-his antigen and αvβ1-his, αvβ3-his, αvβ5-his, αvβ6-his, α2bβ3-his, α5β1-his, α8β1-his (all purchased from ACRO) were diluted in PBSAJ (PBS containing 0.1% BSA, 1 mM Mg ²⁺ and 1 mM Ca ²⁺ ) at 100 ng/100 μl/well overnight. The next day, after washing the plate five times, incubate with 20nM anti-αvβ8 antibodies (Antibody 2, Antibody 4, Antibody 9, Antibody 13, Antibody 16, Antibody 21, Antibody 22, Antibody 23, Antibody 24, Antibody 25, Antibody 27. Antibody 28) diluted in PBSAJ. After washing the plate 8 times, incubate with anti-Kappa HRP for 1 h. After washing the plate 8 times, the color was developed and the reaction was terminated, and the microplate reader was used for reading (the results are shown in Table 3).

Table 3: Binding of anti-αvβ8 antibodies to integrin protein by ELISA

3.2 Binding ability of anti-αvβ8 antibody to cell surface antigen.

CHO cells overexpressing human αvβ8 (CHO-αvβ8 cells) were generated by transfection of the pCMV vector carrying the human αvβ8 cDNA. CHO-αvβ8 cells (0.5×10 ⁶ cells) were mixed with 20 nM anti-human αvβ8 antibody in PBSJ and incubated on ice for 40 minutes. Cells were then washed 3 times and incubated with anti-Fc PE fluorescent secondary antibody in PBS (with 0.2% BSA) for 25 minutes on ice. Cells were washed 3 times and flow cytometric analysis was performed on the Accuri C6 system (BD Biosciences) and the results are shown in Figure 2. The results showed that all anti-human αvβ8 antibodies ( antibodies 2, 4, 9, 13, 16, 21-25, 27 and 28) could significantly bind to the αvβ8 protein on CHO cells.

Example 4: Anti-αvβ8 antibodies block the binding of αvβ8 to its ligand L-TGFβ

CHO-αvβ8 cells (0.5×10 ⁶ cells) were incubated with 4nM L-TGFβ and 10nM, 30nM anti-αvβ8 antibodies (antibody 9, antibody 16) and control antibodies C6D4 and 264RAD, respectively, and the buffer used was PBSAJ . After 40 minutes of incubation, wash three times, and then incubate with anti-Fc PE fluorescent secondary antibody for 25 minutes. Cells were washed 3 times and flow cytometric analysis was performed on the Accuri C6 system (BD Biosciences) and the results are shown in Figure 3 . The results showed that antibody 9 and antibody 16 could block the binding of αvβ8 to its ligand L-TGFβ, and the blocking effect increased with the increase of concentration, and the binding of L-TGFβ and αvβ8 was almost completely blocked at the concentration of 30 nM.

Example 5: Determination of Affinity of Anti-Human αvβ8 Antibody by BIACORE

The affinity of the anti-human αvβ8 antibody was determined according to the instructions of BIACORE. The specific process was to first bind the antibody with a probe, and then detect the binding and dissociation of αvβ8, αvβ6 and αvβ3 at 40nM and 10nM, so as to calculate the exact affinity of the corresponding antibody. There are two buffer systems used, one is HBS-P ⁺ running buffer (containing 100 μg/mL BSA, without 1 mM Mg ²⁺ and 1 mM Ca ²⁺ ). The other is HBS-P running buffer (containing 100 μg/mL BSA, 1 mM Mg ²⁺ and 1 mM Ca ²⁺ ), and the results are shown in Table 4. The results showed that the affinity constants of antibody 9 and αvβ8 and αvβ3 were 26.3nM and 22nM, respectively, and the affinities of antibody 16 and αvβ8, αvβ6 and αvβ3 were 14.6nM, 31.9nM and 5.18nM, respectively.

Table 4: Precise determination of the affinity of anti-human αvβ8 antibody with BIACORE ("-" in the table means no binding)

Example 6: Detection of cell biological activity of anti-αvβ8 antibody

The TGF[beta] biological activity assay was used here, and the method referenced was Abe et al., Anal. Biochem. 216:276-284 (1994). In organisms, L-TGFβ is assembled with GARP proteins through disulfide bonds and displayed on the cell membrane through GARP. When αvβ8 on the cell membrane binds to the RGDL site of L-TGFβ, through mechanical action, the active TGFβ is released from L-TGFβ, thereby activating downstream signaling pathways. The cDNAs of GARP and L-TGFβ (purchased from Yiqiao Shenzhou) were transferred into CHO to obtain CHO cells (named CHO-GARP-L-TGFβ) overexpressing GARP and L-TGFβ. The pGL6-PAI-1-lufiferas-reporter plasmid (purchased from Biyuntian) was electroporated into MLEC cells. In a 96-well plate, CHO-αvβ8 cells (100,000/100 μL well) and CHO-GARP-L-TGFβ cells (100,000/100 μL well) were co-plated and incubated with 100nM and 300nM anti-human αvβ8 antibodies, and 100 nM of C6D4, 100 nM and 300 nM of 264RAD, and supplemented with (1 mM Mg ²⁺ and 1 mM Ca ²⁺ ) in the medium. Then incubate for 48 hours. After that, MLECs were plated at 50,000/100 μL/well, and 100 μL of the cell culture medium supernatant after 48 hours was taken, added to the MLEC cells, and incubated for another 18 hours. 50 μL of fluorescent reactant (ONE-Glo ^™ Luciferase Assay System, purchased from Promega) was added to each well, and read with a microplate reader. The results are shown in Figure 4. The results showed that antibody 9 and antibody 16 significantly blocked the release of TGFβ at the concentrations of 100 nM and 300 nM, thereby inhibiting its downstream signaling.

All publications and patent documents cited herein are incorporated by reference as if each such publication or document were specifically and individually indicated to be incorporated by reference. Citation of the above publications and patent documents does not constitute an admission that any of the above is pertinent prior art, nor does it constitute an admission of its content or date. Now that the invention has been described in the written specification, those skilled in the art will recognize that the invention may be practiced in a variety of embodiments, and the foregoing specification and examples are intended to illustrate rather than limit the claims of the invention.

Claims (18)

1. An antibody or antigen-binding fragment, characterized in that the antibody or antigen-binding fragment specifically binds to αvβ8, and the antibody or antigen-binding fragment comprises:

   (a) VH CDR1, it comprises the amino acid sequence shown in SEQ ID NO:5,

   (b) VH CDR2, it comprises the amino acid sequence shown in SEQ ID NO:6,

   (c) VH CDR3, it comprises the amino acid sequence shown in SEQ ID NO:7,

   (d) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:44,

   (e) a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 45, and/or

   (f) VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:46.
2. The antibody or antigen-binding fragment of claim 1, wherein the VH CDR3 comprises any of the amino acid sequences shown in SEQ ID NOs: 8-25.
3. An antibody or antigen-binding fragment, characterized in that the antibody or antigen-binding fragment specifically binds to αvβ8, and the antibody or antigen-binding fragment comprises:

   (a) VH CDR1, it comprises the amino acid sequence shown in SEQ ID NO:5,

   (b) VH CDR2, it comprises the amino acid sequence shown in SEQ ID NO:6,

   (c) a VH CDR3 comprising any of the amino acid sequences shown in SEQ ID NOs: 8-25,

   (d) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:44,

   (e) a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and

   (f) VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:46.
4. An antibody or antigen-binding fragment, characterized in that the antibody or antigen-binding fragment comprises a VH CDR1 as shown in SEQ ID NO:5, a VH CDR1 as shown in SEQ ID NO:6, such as SEQ ID NO:12 or the VH CDR3 shown in 14, the VL CDR1 shown in SEQ ID NO:44, the VL CDR2 shown in SEQ ID NO:45, and the VL CDR3 shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:8, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:9, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:10, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:11, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:13, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:15, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:16, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:17, as in SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:18, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:19, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:20, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:21, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:22, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:23, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:24, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46; or

   The antibody or antigen-binding fragment comprises a VH CDR1 as set forth in SEQ ID NO:5, a VH CDR1 as set forth in SEQ ID NO:6, a VH CDR3 as set forth in SEQ ID NO:25, such as SEQ ID NO:44 VL CDR1 as shown, VL CDR2 as shown in SEQ ID NO:45, and VL CDR3 as shown in SEQ ID NO:46.
5. The antibody or antigen-binding fragment of any one of claims 1-4, wherein the heavy chain variable region of the antibody or antigen-binding fragment comprises any of the amino acid sequences shown in SEQ ID NOs: 26-43 , or an amino acid sequence having at least 90% sequence homology with any of the amino acid sequences shown in SEQ ID NOs: 26-43; and/or the light chain variable region of the antibody or antigen-binding fragment comprises SEQ ID NO: The amino acid sequence shown in 4, or the amino acid sequence with at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 4.
6. An antibody or antigen-binding fragment, wherein the variable region of the heavy chain of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:26, and the variable region of the light chain comprises the amino acid sequence shown in SEQ ID NO:4 the amino acid sequence of ; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 28, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:34, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:35, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:36, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:40, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:42, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4; or

   The heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:4.
7. The antibody or antigen-binding fragment of any one of claims 1-6, wherein the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 47, or the same as SEQ ID NO: 47. The amino acid sequence shown in NO: 47 has at least 90% sequence homology; and/or the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 48, or the same as SEQ ID NO: 48. The amino acid sequence shown in ID NO: 48 has at least 90% sequence homology.
8. The antibody or antigen-binding fragment of claim 7, wherein the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 47, and the light weight of the antibody or antigen-binding fragment The chain constant region comprises the amino acid sequence shown in SEQ ID NO:48.
9. The antibody or antigen-binding fragment of any one of claims 1-6, wherein the heavy chain of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 49 or 50, or the same as SEQ ID NO: 49 or 50. The amino acid sequence shown in NO: 49 or 50 has at least 90% sequence homology; and/or the light chain of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 3, or the same as SEQ ID NO: 3. The amino acid sequence shown in ID NO: 3 has at least 90% sequence homology.
10. An antibody or antigen-binding fragment, wherein the heavy chain of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:49, and the light chain of the antibody or antigen-binding fragment comprises SEQ ID NO:3 the amino acid sequence shown; or

    The heavy chain of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:49, and the light chain of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:3.
11. The antibody or antigen-binding fragment of any one of claims 1-10, wherein the antibody or antigen-binding fragment specifically binds to αvβ3 and/or αvβ6.
12. A composition, characterized in that the composition comprises the antibody or antigen-binding fragment of any one of claims 1-11, and a pharmaceutically acceptable carrier.
13. A nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1-11.
14. A vector or host cell comprising the nucleic acid molecule of claim 13.
15. Use of the antibody or antigen-binding fragment of any one of claims 1-11, or the composition of claim 12 in the preparation of a medicament for the treatment or amelioration of TGFβ-related diseases, or in the preparation of a diagnosis Use of the kit for TGFβ-related diseases.
16. A kit for diagnosing TGFβ-related diseases, characterized by comprising the antibody or antigen-binding fragment of any one of claims 1-11.
17. A method of treating or ameliorating a TGFβ-related disease in a patient in need thereof, the method comprising administering to the patient an effective dose of the antibody or antigen-binding fragment of any one of claims 1-11, or claim 12 the described composition.
18. The use of claim 15, the kit of claim 16, or the method of claim 17, wherein the TGFβ-related disease comprises cancer or autoimmune disease, and the cancer comprises hematological cancer and solid tumors.

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